Overall goals of the proposed research are two in number: (1) to continue the development of mass spectrometry instrumentation, software, and methodology for the identification and sequence analysis of proteins in complex mixtures at the attomole/femtomole level and (2) to employ this technology to solve structural problems at the cutting edge of biological research. Specific aims include: (1) development of automated mass spectrometry instrumentation for the differential display and quantitation of proteins up- or down-regulated in diseased vs healthy cells, organelles, or tissues; (2) continued development of technology for the selective sequence analysis of phosphorylated peptides and proteins; (3) elucidation of the histone code, the pattern of post-translational modifications on the n-terminal tails of histone proteins that regulate transcription, gene silencing, DNA damage repair, chromosome condensation in apoptosis, chromosome segregation during mitosis,etc; (4) development of technology that facilitates systematic identification of all physiologically relevant substrates and their phosphorylation sites for particular protein kinases on a genome wide scale; (5) identification of protein-protein interaction partners of telomerase, the human reverse transcriptase that maintains chromosome length and allows cancer cells to become immortal; (6) identification of proteins differentially expressed in plasma that can be used to predict onset of myocardial infarction; (7) identification of proteins in cerebrospinal fluid that can be used to predict onset of migraine headaches or to serve as targets for therapeutic intervention; (8) identification of proteins differentially expressed by Pseudomonas aeruginosa when grown under anaerobic (cystic fibrosis) vs aerobic conditions; (9) identification of proteins differentially expressed in the hemolymph of mosquitos before and after infection with malaria parasites; (10) identification of proteins that function as diagnostic markers or druggable targets for acute myeloid leukemias; and (11) identification of phosphorylated proteins in the nucleus and mitochondria of HEK cells before and after treatment with several different drugs.